Ntu 1: Verbascoside Tiv Thaiv Pancreatic -Cells Tawm tsam ER-Stress
Mar 05, 2022
Hu rau: Audrey Hu Whatsapp / hp: 0086 13880143964 Email:audrey.hu@wecistanche.com
Alessandra Galli 1, ntxiv rau, Paola Marciani 1, ntxiv rau, Algerta Marku 1, Silvia Ghislanzoni 1, Federico Bertuzzi 2, Rafaella Rossi 3, Alessia Di Giancamillo 3, Michela Castagna 1 thiab Carla Perego 1, *
Abstract:
Cov pov thawj ntawm kev kis mob loj heev qhia tau hais tias kev noj zaub mov nplua nuj nyob hauv polyphenols tiv thaiv kev txhim kho ntshav qab zib hom 2. Lub phenylethanoid glycosideverbascoside / acteoside, ib qho chaw cog qoob loo polyphenolic, muaj ntau yam khoom siv lom neeg xws li muaj zog antioxidant, tiv thaiv kab mob thiab kev tiv thaiv kab mob. Lub hom phiaj ntawm qhov kev tshawb fawb no yog los ntsuas qhov ua tau tshwm sim ntawmlus verbascosidentawm pancreatic-cells, lub hom phiaj yeej tsis tau sim ua ntej. Nas thiab tib neeg-cell tau incubated nroglus verbascoside(0.8–16 uM) txog li tsib hnub thiab kev sib xyaw ua ke ntawm biochemical thiab imaging cov tswv yim tau siv los ntsuam xyuas -cell ciaj sia taus thiab ua haujlwm nyob rau hauv ib txwm lossis endoplasmic reticulum (ER)-kev ntxhov siab inducing tej yam kev mob. Peb pom ib koob tshuaj tiv thaiv kab mob ntawmlus verbascosidetiv thaiv oxidative kev nyuaj siab nyob rau hauv clonal thiab tib neeg-cells. Cov kev tshawb fawb Mechanistic tau qhia tias cov polyphenol tiv thaiv -cells tiv thaiv ER-kev ntxhov siab kho kom haum xeeb tsis ua haujlwm, hloov pauv kev ua kom cov protein kinase RNA-zoo li endoplasmic reticulum kinase (PERK) ceg ntawm cov lus teb tsis muaj protein ntau thiab txhawb mitochondrial dynamics. Raws li qhov tshwm sim, nce kev vam meej, kev ua haujlwm ntawm mitochondrial thiab cov ntsiab lus insulin tau kuaj pom hauv cov hlwb. Cov kev tshawb fawb no muab pov thawj tiaslus verbascosideboosts lub peev xwm ntawm -cells los tiv nrog ER-kev nyuaj siab, ib qho tseem ceeb contributor ntawm -cell dysfunction thiab tsis ua hauj lwm nyob rau hauv cov mob ntshav qab zib thiab pab txhawb lub peev xwm kho mob ntawm verbascoside nyob rau hauv cov ntshav qab zib.
Ntsiab lus:
verbascoside; polyphenols; cov hlwb tsim cov tshuaj insulin; ntshav qab zib; UPR; oxidative kev nyuaj siab; ER-kev nyuaj siab; PERK; anti-inflammatory; mitochondria cov tsos mob
1. Taw qhia
Mob ntshav qab zib yog ib qho mob ntev uas cuam tshuam ntau pua lab tus tib neeg [1]. Kev sib txawv etiology characterizes hom 1 (T1D) thiab hom 2 mob ntshav qab zib mellitus (T2D) ob qho tib si featuring tsis muaj insulin [2]. Insulin tswj cov ntshav qabzib concentration stimulating qabzib uptake hauv cov leeg nqaij thiab cov rog thiab modulating daim siab cov piam thaj metabolism. Muaj cov khoom noj muaj txiaj ntsig, cov tshuaj hormones, thiab cov khoom siv neural tswj hwm pancreatic insulin secretion thiab tswj cov ntshav qabzib ntau hauv lub cev [3-5]. Yog li, -cell dysfunction ua rau cov ntshav qab zib ua cim los ntawm kev yoo mov hyperglycemia.
T2D yog ib qho mob hnyav heev uas cov tshuaj insulin tsis kam thiab -cells ua haujlwm tsis raug cuam tshuam nrog ua ke thiab cov pov thawj tsis ntev los no tau nce kev txaus siab rau lub luag haujlwm tseem ceeb ntawm -cells rau cov xwm txheej hyperglycemic [6,7]. Hauv cov neeg uas tsis muaj ntshav qab zib mellitus - cov cell them nyiaj rau insulin tsis kam nrog kev tso tawm ntawm cov tshuaj hormones. Txawm li cas los xij, hauv qee qhov kev kawm, raws li lawv cov xwm txheej tsis zoo, insulinemia poob thiab qib qabzib nce mus rau hyperglycemia [8,9].
Glucose yog qhov tseem ceeb tshaj plaws modulator ntawm -cell functions. Glucose stimulation cuam tshuam rau kev tswj cov noob thiab kev qhia ntawm cov proteins koom nrog ntau lub zog ntawm tes xws li glycolysis, insulin synthesis thiab secretion [10]. Glucose-induced insulin secretion cia siab rau oxidative metabolism los tsim adenosine triphosphate (ATP) thiab qib qis ntawm cov pa oxygen reactive (ROS) physiologically tsim. Txawm li cas los xij, -cells tsis zoo heev nrog cov enzymes scavenger thiab qhov tsis muaj zog no ua rau lawv raug oxidative kev nyuaj siab [11,12]. Raws li oxidative kev nyuaj siab nce -cells insulin ntau lawm poob, thiab -cells tsim cytokines uas ignite cell puas los ntawm o thiab apoptosis [13]. Interestingly, txawm hais tias qhov txo qis-cells loj tau dhau los ua rau kev tuag ntawm tes, kev tshawb fawb tsis ntev los no qhia txog lub luag haujlwm tseem ceeb ntawm cell dediferentiation [14,15]. Nws tau dhau los ua qhov tseeb tias ntau theem tsav mus rau T2D. Metabolic thiab oxidative insults ua rau endoplasmic reticulum (ER) kev nyuaj siab uas ua rau poob ntawm insulin synthesis thiab secretion, cov txheej txheem o ua raws li kev tso tawm ntawm cytokines thiab -cells poob yuav tshwm sim los ntawm apoptosis thiab dediferentiation. Qhov kev paub no yog qhov tseem ceeb txhawm rau txhawm rau txhim kho cov tswv yim tsim nyog ntawm kev kho mob, txij li oxidative kev nyuaj siab thiab o tuaj yeem tiv thaiv, thiab cov yas ntawm cov kab mob pancreatic tso cai rau muaj peev xwm rov qab cov qia hlwb rau -cells raws li tau pom hauv nas [16].
Tsis ntev los no, qhov tsis zoo ntawm -cells rau oxidative kev nyuaj siab tau ua tiav kev siv cov khoom noj antioxidants los tiv thaiv ntshav qab zib [17]. Ntawm cov khoom noj antioxidant zoo tshaj plaws, txiv roj roj yog paub txog nws cov txiaj ntsig zoo txij li cov neeg Greek thaum ub. Cov roj txiv roj muaj cov txiaj ntsig zoo ntawm mono-unsaturated fatty acids (MUFA) thiab ntau cov polyphenols xws li tyrosol, hydroxytyrosol, oleuropein, thiab verbascoside.
Verbascoside, tseem hu ua acteoside, yog phenylethanoid glycoside rho tawm los ntawm Olea Europe, cov nroj tsuag ntawm Verbascum hom, thiab 23 lwm cov tsev neeg cog [18–20]. Nws tseem tuaj yeem tau txais los ntawm cov roj txiv roj los ntawm cov khoom lag luam (nws yog enriched nyob rau hauv cov txiv ntseej-mills cov dej khib nyiab tau los ntawm cov txiv ntseej ua cov txiv ntseej) los yog tsim los ntawm cov txheej txheem metabolic thiab hluavtaws biology [21].
Tsis muaj ntaub ntawv qhia txog verbascoside bioavailability hauv tib neeg, tseem. Cov kev tshawb fawb tau ua rau nas, SKBR3 thiab Caco-2 hlwb qhia tias nws yuav ua tau rau cov uas tsis yog-metabolized verbascoside kom hla txoj hnyuv, ncig hauv cov ntshav plasma, thiab siv cov tshuaj antioxidant [20,22,23]. Tsis zoo li feem ntau ntawm cov nroj tsuag polyphenols, verbascoside feem ntau ua rau cov hlwb los ntawm kev hloov pauv ntawm cov noob hloov pauv ntawm ntau yam enzymes thiab cov cai tswj hwm, nrog rau cov tshuaj tiv thaiv antioxidant thiab tiv thaiv kab mob [24-30].
Txawm hais tias muaj ntau qhov txiaj ntsig zoo ntawm verbascoside rau tib neeg kev noj qab haus huv tau paub, tsis muaj cov ntaub ntawv hais txog nws cov txiaj ntsig ntawm cov kab mob pancreatic. Txij li thaum oxidative kev nyuaj siab thiab o yog nyob rau hauv lub hauv paus ntawm T2D pathogenesis, peb soj ntsuam seb verbascoside kev kho mob yuav txhim kho -cells viability thiab
ua haujlwm nyob rau hauv ER-kev ntxhov siab inducing tej yam kev mob thiab peb characterized lub molecular mechanisms ntawm nws cov kev ua. Peb cov ntaub ntawv qhia tau hais tias verbascoside tiv thaiv -cells oxidative kev nyuaj siab thiab inflammatory modulating ua kom lub unfolded protein teb thiab txhawb mitochondrial dynamics, yog li ua rau kom - cell viability thiab insulin cov ntsiab lus.
2. Cov ntaub ntawv thiab cov txheej txheem
2.1. Cells Culture thiab Materials
Mouse tc3 hlwb (kev ua siab zoo muab los ntawm Prof. Hanahan—Department of Biochemistry thiab Biophysics, University of California, San Francisco, CA [31]) tau coj mus kuaj hauv RPMI 1640 nruab nrab (Euroclone SpA, ECB90 0, Pero MI, Ltalis) ntxiv nrog 10 feem pua (v/v) kub-inactivated fetal calf serum (Euroclone SpA, ECS0180L, Pero MI, Ltalis), 1 feem pua (v/v) penicillin-streptomycin (EuroClone SpA, ECB3001D, Pero MI, Ltalis) thiab 1 feem pua (v/v) L-glutamine (EuroClone SpA, ECB300D, Pero MI, Ltalis). Tib neeg islets ntawm Langerhans raug cais nyob rau hauv Milan (Niguarda Ca' Granda) los ntawm cadaveric multiorgan pub dawb raws li cov txheej txheem piav qhia los ntawm Ricordi li al. [32]; Lawv tau coj mus kuaj hauv RPMI kab lis kev cai nruab nrab uas muaj 5.5 mmol / L qabzib, 10 feem pua ntawm cov menyuam yaus hauv plab thaum tshav kub kub, 0.7 mM Glutamine, 50 units / mL penicillin thiab 50 ug / mL streptomycin (EuroClone, SpA, Pero MI, Ltalis). Plaub qhov sib txawv islets npaj tau siv, islets purity yog 80 ± 10 feem pua. Kev cais tawm ntawm Islet thiab islet kev tshawb fawb tau pom zoo los ntawm Pawg Neeg Saib Xyuas Kev Ncaj Ncees ntawm Niguarda Ca' Granda tsev kho mob hauv Milan (11.12.2009). tc3 hlwb raug kho nrog 0.8, 1.6, thiab 16 uM verbascoside (Carbosynth, OV08034, Compton, UK), cafeic acid thiab hydroxytyrosol (zoo khoom plig ntawm Prof. Dell'Agli, Department of Pharmacological thiab Biomolecular Sciences, Universita degli Studi di Milano, Milan, Ltalis) ua tiav RPMI nruab nrab rau 5 hnub, thaum tib neeg islets nrog 16 uM verbascoside. Methanol / ethanol kho hlwb tau siv los tswj. Yuav kom induce oxidative kev nyuaj siab, hlwb raug kho nrog H2O2 (Sigma Aldrich, H1009, St. Louis MO, USA) 500 uM nyob rau hauv tag nrho RPMI nruab nrab rau 20 min ua ntej tsom xam, thaum kho nrog tunicamycin 2 ug/mL (T7765, Sigma Aldrich. ) rau 7 h tau ua los txhawb ER kev ntxhov siab.
2.2. Kev kuaj pom ntawm Cell Viability los ntawm MTT Test
Cells raug plated nyob rau hauv 96 multi-zoo daim hlau, thiab tsib hnub tom qab kev kho mob verbascoside lawv tau incubated nrog 0.5 mg/mL MTT (3-(4,5-dimethyltiazol-2- }yl)-2,5-diphenyltetrazolium bromide) (Sigma-Aldrich, M5655, St. Louis, MO, USA) rau 4 h hauv huab cua huab cua uas muaj 5 feem pua ntawm CO2 ntawm 37 oC. Tom qab incubation, hlwb tau maj mam resuspended nyob rau hauv 100 uL DMSO (Euro-Clone SpA, BK12611S, Pero MI, Ltalis) thiab lub absorbance ntawm 540 nm tau kuaj pom nrog ib tug microplate nyeem ntawv (Benchmark, microplate nyeem, Bio-Rad Laboratories, Hercules CA, USA) [33]. Cov kev sim tau ua nyob rau hauv triplicate thiab cov ntaub ntawv tau nthuav tawm raws li kev nce ntxiv ntawm cov qauv tswj.
2.3. Kev kuaj pom ntawm Cell Tuag los ntawm Flow Cytometry
Tsib hnub tom qab incubation nrog 16 uM verbascoside, cov -cells tau detached los ntawm 7 min incubation nrog trypsin / EDTA, sau thiab centrifuged; cov pellet tau maj mam resuspended hauv phosphate bufer saline tsawg ntsev. Cells tau stained nrog MuseTM suav thiab muaj peev xwm reagent (Millipore, MCH100102, Burlington MA, USA) ua raws li cov chaw tsim khoom raws tu qauv thiab soj ntsuam los ntawm cov cytometry ntws. Cov kev sim tau ua nyob rau hauv triplicate thiab cov ntaub ntawv tau nthuav tawm raws li feem pua ntawm cov hlwb tuag tshaj tag nrho.
2.4. ROS tiam
Intracellular ROS raug soj ntsuam nrog DCFDA (2/,7/-dichlorofouorescein diacetate) (Sigma Aldrich, D6883, St. Louis, MO, USA), ib qho kev sojntsuam membrane permeable uas dhau los ua fluorescent thaum khi rau ROS [34]. tc3 hlwb tau pre-loaded nrog 15 uM DCFDA hauv Krebs-Ringer bufer (125 mM NaCl, 5 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 25 mM HEPES-NaOH pH 7.4 thiab 2 mM CaCl2)
ntxiv nrog 11 mM qabzib rau 1 teev ntawm 37 oC. Cov ntsiab lus ROS tau kuaj pom rau 30 feeb ob qho tib si hauv basal thiab kev ntxhov siab nrog lub microplate nyeem ntawv (485/528 nm Ex/Em) (TECAN Infinite⑧ F500, Tecan Group Ltd. Männedorf, Switzerland). Tus nqi nruab nrab thiab tus qauv sib txawv yog nyob ntawm peb qhov kev sim ywj pheej.
2.5. Western Blotting
tc3 hlwb raug sau thiab solubilized nyob rau hauv RIPA bufer (150 mM NaCl, 50 mM Tris HCl pH 7.6, 1 mM EDTA, 1 feem pua TERGITOLw NP40, 0.5 feem pua deoxycholate) ntxiv nrog aprotinin (Sigma Aldrich, St. Louis, MO, USA), PMSF (Sigma Aldrich, 10837091001, St. Louis, MO, USA) thiab Roche inhibitors (Sigma Aldrich, 5892953001, St. Louis, MO, USA) rau 40 min ntawm 4 oC. Protein concentration tau txiav txim siab los ntawm Bradford assay [35] los ntawm kev siv Bradford Reagent (Sigma Aldrich, B6916, St. Louis, MO, USA), 30 ug ntawm cov protein tau daws los ntawm 10 feem pua SDS-PAGE thiab xa mus rau nitrocellulose membranes (Millipore, Burlington. MA, USA). Cov tshuaj tiv thaiv thawj zaug tau thov rau 2 teev hauv kev thaiv cov bufer nrog 5 feem pua cov mis tsis muaj rog lossis 5 feem pua BSA daws; Cov tshuaj tiv thaiv kab mob hauv qab no tau siv: nas anti- -actin (Novus International Inc., NB600501, St. Louis, MO, USA), nas anti-acrolein (Abcam, ab48501, Cambridge, UK), nas anti-BIP (zoo khoom plig ntawm Prof. Borgese Nica, lub koom haum ntawm Neuroscience, CNR, Milan, Ltalis), luav anti-HNE ( -diagnostic International Inc., HNE11S, San Antonio, TX, USA), nas anti-HSP70 (Enzo Life Sciences Inc. ., C92F3A-5, Farmingdale, NY, USA), luav anti-phospho-IKB (Ser32) (Cell Signaling Technology Inc., 3033, Danvers, MA, USA), nas anti-IKB (Cell Signaling Technology Inc. ., 4814, Danvers, MA, USA), luav anti-phospho-NFKB p65 (Ser 536) (Cell Signaling Technology Inc., 3033, Danvers, MA, USA), luav anti-NFkB p65 (Cell Signaling Technology Inc., 8242, Danvers MA, USA), yaj anti-SOD1 (Merck, KGaA, Darmstadt, Germania), luav anti-PERK (Cell Signaling Technology Inc., 3192, Danvers, MA, USA), luav anti-eIF2 (Cell Signaling Technology Inc., 5324, Danvers, MA, USA) thiab luav anti-P-eIF2 (Ser 51) (Cell Signaling Technology Inc., 3597, Danvers, MA, USA). Cov tshuaj tiv thaiv thib ob HRP-conjugated (Dako Agilent, Santa Clara, CA, USA) tau siv ntawm 1: 5000 dilution. Cov Proteins tau kuaj pom los ntawm kev siv ECL kev kuaj xyuas qhov system (Euro-Clone SpA, Pero MI, Ltalis) los ntawm kev siv Odyssey Fc Image system (LI-COR Biotechnology GmbH, Bad Homburg, Lub teb chaws Yelemees) thiab band ceev tau txiav txim siab los ntawm Image Studiow Lite software (LI -COR Biosciences, Lincoln, NE, USA) [36]. Cov kev sim tau ua nyob rau hauv triplicate thiab cov ntaub ntawv tau nthuav tawm raws li kev nce ntxiv ntawm cov qauv tswj.
2.6. Mitochondrial Membrane muaj peev xwm
tc3 hlwb thiab tib neeg islets ntawm Langerhans tau incubated nrog 100 nM MitoSpyw Txiv kab ntxwv CMTMRos (BioLegend, 424803, Campoverde Srl, Milan, Ltalis) los yog 100 uM MitoSpyw Green FM (BioLegend, 424805, Campoverde Srl, 30 Milan, Ltalis) oC; Fluorescence intensities raug kuaj pom nrog tus nyeem ntawv microplate TECAN Innite⑧ F500 (551/576 nm Ex/Em rau MitoSpyw Txiv kab ntxwv CMTMRos; 490/516 nm Ex/Em rau MitoSpyw Green) (TECAN Infinite, Ltd. Cells raug incubated nrog 500 uM H2O2 rau 20 min thiab qhov fluorescence siv tau raug kuaj pom raws li tau piav qhia dhau los. Tus nqi nruab nrab thiab tus qauv sib txawv tau ua raws li peb qhov kev sim sib txawv.
2.7. Mitochondrial Morphology thiab Dynamics
tc3 hlwb tau pre-loaded nrog 100 nM MitoSpyw Txiv kab ntxwv CMTMRos (BioLegend, 424803, Campoverde Srl, Milan, Ltalis) hauv 11 mM qabzib Krebs-Ringer bufer ntawm 37 oC rau 30 min. Cov qauv tau muab tso rau hauv chav ntsuas duab thiab cov duab random tau pom los ntawm kev siv rhodan lim ntawm Axio Observer Z1 microscope (Zeiss, Oberkochen Lub Tebchaws Yelemees). Txhawm rau ntsuas mitochondrial morphology, cov ntsuas hauv qab no tau txheeb xyuas los ntawm kev siv ImageJ particle analyzer software: cheeb tsam (um2), ncig (4mArea2 / Perimeter2) thiab Feret qhov siab tshaj plaws txoj kab uas hla (um) [37].
Rau lub sij hawm-lapse thwmsim, ib leeg-cell duab tau ua nyob rau ntawm 1 thav duab ib ob rau 30 s nyob rau hauv kev tswj los yog oxidative kev nyuaj siab. Txhawm rau ntsuas qhov mitochondrial cumulative nrug (um2), cov duab tau raug kho thawj zaug rau cov duab bleaching, tom qab ntawd cov yeeb yaj kiab raug tshuaj xyuas los ntawm kev siv cov duab-Pro Plus Plug-in uas twb muaj lawm (cov khoom taug qab) (Media Cybernetics, Rockville, MD, USA). Mus txog kaum ob
cov hlwb tau pom nyob rau hauv peb qhov kev sim ywj pheej thiab cov ntaub ntawv tau nthuav tawm raws li qhov tseem ceeb thiab tus qauv sib txawv.
2.8. Insulin secretion
Tib neeg cov islets cais ntawm Langerhans tau muab noob rau hauv 96-zoo phaj ntawm qhov ntom ntawm 20 islets ntawm ib qhov dej thiab, tom qab 5 hnub ntawm kev kho mob, cov ntsiab lus ntawm insulin thiab tso tawm tau ntsuas hauv basal (3.3 mM qabzib) thiab txhawb nqa (16.7 mM qabzib). ) cov xwm txheej los ntawm ELISA immunoassay (Mercodia, 10-1113-01, Uppsala, Sweden).
2.9. Kev Tshawb Fawb Txog Kev Tshawb Fawb
Txhua qhov kev txheeb xyuas txheeb cais tau ua nrog GraphPad Prism 8.0 ntawm cov khoom siv roj ntsha ywj pheej Txhais tau hais tias ntawm ob pab pawg raug soj ntsuam los ntawm kev siv ob-tailed Student's t-test thiab p-value < 0.05="" raug="" coj="" los="" ua="" pov="" thawj="" ntawm="" qhov="" tseem="" ceeb.="" txhais="" tau="" hais="" tias="" ntawm="" peb="" lossis="" ntau="" pawg="" tau="" muab="" piv="" los="" ntawm="" kev="" txheeb="" xyuas="" qhov="" sib="" txawv="" (anova),="" tom="" qab="" los="" ntawm="" ntau="" qhov="" kev="" sib="" piv="" tom="" qab-hoc="" (tukey's)="" .="" qhov="" kev="" ntsuas="" ntsuas="" tau="" siv,="" qhov="" tseeb="" p="" qhov="" tseeb="" thiab="" tus="" lej="" ntawm="" tus="" lej="" replica="" (n)="" tau="" qhia="" nyob="" rau="" hauv="" tus="" kheej="" cov="" duab="" dab="" neeg.="" cov="" ntaub="" ntawv="" yuam="" kev="" hauv="" cov="" duab="" qhia="" qhov="" txhais="" tau="" tias="" ±="" sd="" lossis="" qhov="" nruab="" nrab="" ±="" se,="" raws="" li="">
3. Cov txiaj ntsig
3.1. Verbascoside Txhim Kho -Cells Viability
Txij li thaum tsis muaj cov ntaub ntawv ntawm verbascoside efect ntawm -cells, peb tau ua MTT xeem los soj ntsuam cov cell viability thiab raws li qhia nyob rau hauv daim duab 1A, ib tug zoo koob tshuaj-dependent qauv tau pom. Li no, rau cov kev tshawb fawb ntxiv, peb tau xaiv 16 uM concentration uas tau txheeb xyuas qhov ua tau zoo los txhim kho -cells viability.By flow cytometry peb tau kawm txog qhov cuam tshuam ntawm tsib hnub incubation ntawm verbascoside ntawm -cell ciaj sia nyob rau hauv basal tej yam kev mob thiab tom qab 2{{21} } min ntawm kev kho ua ntej nrog 500 uM H2O2. Nyob rau hauv cov xwm txheej basal, verbascoside tsis cuam tshuam rau cell ciaj sia, qhia tias qhov nce-cell viability pom nyob rau hauv MTT kev soj ntsuam yuav yog vim kev txhim kho mitochondrial kev ua. Whereas, tom qab H2O2 raug, 16 uM verbascoside ua ntej kev kho mob tseem ceeb txo oxidative kev nyuaj siab vim -cell tuag (Daim duab 1B, C).Verbascoside yog ib tug complex molecule uas yuav hloov tau los ntawm hydrolyzing enzymes [18]. Txhawm rau kuaj seb verbascoside metabolites puas tuaj yeem ua lub luag haujlwm rau qhov pom kev tiv thaiv, tc3 hlwb tau kho nrog hydroxytyrosol thiab cafeic acids (0.8 thiab 16 uM) rau 5 hnub. Ob leeg metabolites tsis txhim kho cell viability, ntawm qhov tsis sib xws, qhov cuam tshuam cytotoxic, cuam tshuam ntau dua tom qab H2O2 raug, raug kuaj pom rau 16 uM concentration (Daim duab S1). Qhov kev tshawb pom no ua pov thawj tias verbascoside thiab tsis yog nws cov metabolites ua rau muaj kev tiv thaiv zoo rau kev kho H2O2.
3.2. Verbascoside Modulates Redox Homeostasis thiab Exerts Anti-Inflammatory Efect hauv -Cells
Peb thawj zaug tau lees paub hauv tc3 hlwb cov tshuaj tiv thaiv kab mob thiab tshuaj tiv thaiv oxidant ntawm verbascoside pom hauv lwm hom cell [24,29,34]. Lub verbascoside ROS scavenging kev ua si tau soj ntsuam nrog tc3 hlwb sau nrog ROS tshwj xeeb DCFDA (2/,7/-dichlorofouorescein diacetate) cellular permeable sojntsuam. Raws li pom hauv daim duab 2A, qhov txo qis ntawm ROS cov ntsiab lus tau kuaj pom tom qab verbascoside ua ntej kho, ob qho tib si nyob rau hauv basal thiab oxidative kev nyuaj siab.
Western blot tsom xam pom qhov txo qis ntawm oxidative stress markers acrolein thiab 4-hydroxynonenal (HNE) qhia hauv verbascoside-kho hlwb. Tsis tas li ntawd, peb pom qhov nce ntawm superoxide dismutase (SOD1) kev qhia, qhia tias verbascoside exerts nws cov kev ua haujlwm antioxidant tej zaum nrog ob lub tswv yim sib txawv, ncaj qha raws li ROS scavenger thiab tsis ncaj los ntawm inducing kev qhia ntawm antioxidant enzymes (Daim duab 2B, C).
Cov tshuaj tiv thaiv kab mob ntawm verbascoside ntawm -cells tau soj ntsuam los ntawm kev ntsuas qhov ua kom ntawm NFKB txoj hauv kev, qhov tseem ceeb tshaj plaws pro-inflammatory txoj hauv cov hlwb [38]. Los ntawm Western blot tsom xam peb pom ib qho kev txo qis ntawm inhibitor ntawm nuclear factor kappa B (IKB) thiab nuclear factor kappa-light-chain-enhancer (NFKB), thiab qhov txo qis ntawm NFKB phosphorylation hauv cov hlwb ua ntej kho, txhawb kev xav ntawm lub verbascoside effectiveness los txo cellular o (Daim duab 2D, E).
3.3. Verbascoside Modulates lub Unfolded Protein teb ntawm -Cells
Tom qab ntawd peb txheeb xyuas qhov tob ntawm cov txheej txheem molecular los ntawm qhov verbascoside exerts antioxidant thiab anti-inflammatory luag hauj lwm, tsom rau endoplasmic reticulum uas yog tshwm sim los ua ib tug tseem ceeb sensor ntawm metabolic thiab kev nyuaj siab teeb liab nyob rau hauv -cells. Nyob rau hauv cov xwm txheej kev ntxhov siab, lub organelle mounts ib qho homeostatic teb, hu ua unfolded protein teb (UPR), aimed ntawm recovering ER muaj nuj nqi; Txawm li cas los xij, kev ua kom ntau dhau ntawm txoj kev no ua rau apoptosis [39,40].
Cov cim ntawm nce ER kev ntxhov siab yog kev tswj hwm ntawm chaperones proteins thiab ua kom cov lus teb UPR. Western blotting tsom xam ntawm cov hlwb kho nrog verbascoside qhia qhov txo qis ntawm ob chaperonins kub poob siab protein 70 (HSP70) thiab khi immunoglobulin protein (BIP) (Daim duab 3A, B). Tsis tas li ntawd, qhov txo qis ntawm cov protein kinase RNA zoo li ER kinase (PERK) qhia thiab txo cov phosphorylation ntawm nws cov dej ntws qis, qhov kev pib txhais lus eukaryotic 2 (eIF2), tau kuaj pom.
